PROJECT SUMMARY/ABSTRACT Stem cells have critical roles in development and homeostasis; understanding their biology is essential for re- generative medicine, preventing and treating cancer, and developing therapies to combat aging and degenera- tive disease. Important to many stem cell systems are interactions with endocrine factors, for regulating cell cycling, fate-speci cation or differentiation. One such factor is thyroid hormone (TH), which has diverse and often opposing effects across stem cell lineages, and for which defects in signaling lead to mental and physical retardation, peripheral neuropathies, infertility and other disorders. These studies use zebra sh adult pigmen- tation as an innovative and tractable system for elucidating TH functions in differentiation and morphogenesis as well as homeostasis and neoplasia. Adult pigment cells of zebra sh arise from neural crest-derived latent stem or progenitor cells that differentiate during the larval-to-adult transformation, a period that shares similari- ties with late fetal, neonatal and adolescent stages of mammals. Preliminary analyses demonstrated that TH represses the development of black melanophores, homologous to mammalian melanocytes, but promotes the development of yellow/orange xanthophores. Studies in Aim 1 will uncover the cellular bases for these effects, focusing on whether TH speci es alternative fates or promotes terminal differentiation within de ned progenitor lineages, and whether TH modulates morphogenetic behaviors of proliferation, migration or survival. Additional analyses will test for later TH-dependent homeostasis of these lineages and whether TH protects against melanoma onset or progression, as hypothyroidism is signi cantly more prevalent in human melanoma pa- tients than in the general population. Studies in Aim 2 will elucidate molecular mechanisms of TH activities in pigment stem cell lineages by testing roles for canonical ( genomic ) signaling, in which liganded TH receptors directly modulate target gene transcription, and non-canonical mechanisms, in which TH promotes cytoskeletal changes and interacts with a variety of signaling pathways. Additional analyses will test two molecular mecha- nisms that are excellent candidates for mediating TH effects on pigment cells, via hypoxia inducible factor 1? or signaling through the receptor tyrosine kinases Kit and Colony stimulating factor-1 receptor. Experiments in Aims 1 and 2 will employ an innovative suite of approaches including genetic analyses of new mutants, ma- nipulation of TH status using a conditional transgenic line and lineage-speci c transgenic effectors, single cell- fate mapping, and high-resolution time-lapse imaging. Together, these studies will answer several of the most important questions about TH activities in pigmentation while establishing a highly accessible system for ad- dressing TH functions in stem cells more generally.